Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

Abstract

In humans, activated platelets contribute to sepsis complications and to multiple organ failure. In our prospective analytical study of cases of the equine systemic inflammatory response syndrome (SIRS), we adapted a standard human protocol for the measurement of activated platelets and platelet-leukocyte aggregates (PLAs) in equine platelet-leukocyte-rich plasma (PLRP) by flow cytometry, and we investigated the hypothesis that activated platelets and PLAs are increased in clinical cases of SIRS. We included 17 adult horses and ponies fulfilling at least 2 SIRS criteria, and 10 healthy equids as controls. Activation of platelets was determined by increased expression of CD62P on platelets. Activated platelets and PLAs were measured before and after in vitro activation of platelets with collagen. Median expression of CD62P on platelets was significantly increased after activation in the control group: 1.45% (interquartile range [IQR]: 1.08-1.99%) initially versus 8.78% (IQR: 6.79-14.78%, p = 0.002) after activation. The equids with SIRS had significantly more activated platelets and PLAs in native PLRP than controls: CD62P 4.92% (median, IQR: 2.21-12.41%) versus 1.45% in controls (median, IQR: 1.08-1.99%, p = 0.0007), and PLAs 4.16% (median, IQR: 2.50-8.58%) versus 2.95% in controls (median, IQR: 1.57-3.22%, p = 0.048). To our knowledge, increased platelet activation and PLAs have not been demonstrated previously with flow cytometry in clinical cases of equine SIRS.

Keywords: CD62P; P-selectin; equine systemic inflammatory response syndrome; flow cytometry; horses; platelets.

Figure 1.

Cytometric gating protocol. A. Platelet gate (P1) on the basis of the forward (FSC) and side scatter (SSC), threshold FSC 20,000. B. Single-parameter fluorescence histogram of the APC-conjugated polyclonal goat anti-mouse antibody as isotype-negative control for CD41/61 (FL-4: 640 nm, filter 675/25 nm). C. Single-parameter fluorescence histogram gated for APC-conjugated monoclonal mouse anti-sheep antibodies against CD41/61. D. Dotplot representing platelet expression of CD41/61 (y-axis; FL-4; Q1-UL indicating positive events for CD41/61) and expression of CD11a/18 (leukocytes; x-axis; FL-1, 488 nm, filter 530/30 nm; Q1-LR indicating positive events for CD 11a/18); dual-labeled events positive for CD41/61 and CD11a/18 representing 3.5% platelet-leukocyte aggregates in Q1-UR.

Figure 2.

Platelet activation and platelet-leukocyte aggregates (PLAs) in controls (n = 10) and systemic inflammatory response syndrome (SIRS) group (n = 17) in native samples (white boxes) and after in vitro activation with collagen (cross-hatched boxes); boxes are median and IQR, whiskers minimum and maximum. A. Percentage of CD62P-positive platelets; a, p = 0.0007; b, p < 0.0001. B. Percentage of PLAs: c, p = 0.048; d, p = 0.0009; e, p = 0.036.

Figure 3.

Platelet activation and platelet-leukocyte aggregates (PLAs) in the systemic inflammatory response syndrome (SIRS) group (n = 13) depending on outcome in native samples (open circles) and after in vitro activation with collagen (black dots); bar = median. A. Individual percentage of CD62P-positive platelets; a, p = 0.03. B. Individual percentage of PLAs.